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Using Neutron Reflectometry to Determine the Location of Antimicrobial Peptides from Australian Frogs in Model Biological Membranes
Authors
David Fernandez, Thomas Whitwell and Frances Separovic (University of Melbourne), Anton Le Brun, Paramjit Bansal and Michael James (Bragg Institute, ANSTO)
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Figure 1. The antimicrobial peptides maculatin 1.1 (left) and aurein 1.2 (center) and a picture of the green-eyed tree frog, Litoria genimaculata (right). Picture from www.frogsaustralia.net.au |
Antibiotic-resistant ‘superbugs’ such as methicillin-resistant Staphylococcus aureus (MRSA) are become an increasing problem, particularly in places of medical care such as hospitals. It is estimated that approximately 500,000 people a year globally are killed from infections caused by antibiotic-resistant bacteria [1].
Antimicrobial peptides offer one avenue to combat these ‘superbugs’ as the peptide attacks the lipid components of the bacterial membrane making it less possible for the bacteria to build resistance to the antimicrobial peptide. Many Australian frogs have bactericidal skin secretions which contain antimicrobial peptides. We have been studying two synthetic analogues of such peptides: maculatin 1.1 from the green-eyed tree frog and aurein 1.2 from the green and golden bell frog (Figure 1). These peptides are small α-helical antimicrobial peptides and attack the Gram-positive bacterial membrane.
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Figure 2. The scattering length density profile of an anionic phospholipid bilayer of DMPC/DMPG (4:1) on a silica surface before (red line), and after maculatin 1.1 addition (black line). The lower scattering length density of the black line shows that maculatin 1.1 penetrates across the whole bilayer and forms a pore in the membrane. The background image of the lipid bilayer [4] is only a guide to the eye and is not to scale. |
Previous studies using solid-state NMR, quartz-crystal microbalance, florescence microscopy and surface plasmon resonance show that the action of these peptides for disrupting membranes depends on the lipid composition and peptide concentration [2]. In studies of these systems at ANSTO, we used neutron reflectometry to examine the effect of maculatin 1.1 and aurein 1.2 on simple model phospholipid bilayers that mimic the essential charge characteristics of red blood cell (neutral) and bacterial (anionic) membranes.
The membranes were created on silica surfaces using the vesicle deposition technique. The ability to discriminate between hydrogen and its isotope deuterium in neutron scattering makes neutron reflectometry a powerful tool in dissecting the lipid, peptide and solvent components of membranes along the axis perpendicular to the plane of the membrane (the z-axis). In this case hydrogenated peptide and tail deuterated lipids were used. Thus in a D2O solvent the peptide is highlighted and in a H2O solvent the phospholipid tails are highlighted. The experiments were carried out on the Platypus time-of-flight neutron reflectometer at OPAL.
Both antimicrobial peptides were found to bind to both types of model membrane but only surface interactions were observed for the neutral bilayers with little disruption of the bilayer structure. For the anionic bilayers, structural changes were observed upon peptide addition. Upon the addition of maculatin 1.1 to the anionic bilayers, pore-formation along with an increase in lipid tail order and changes in head group (HG) orientation were observed (Figure 2) [3]. When aurein 1.2 bound to the anionic bilayers membrane lysis by the carpet mechanism was observed accompanied by a decrease in lipid tail order.
Using neutron reflectometry to study peptide-lipid interactions gives researchers insights into how peptide insertion affects the structure of phospholipid bilayers and the mechanisms by which antimicrobial peptides work. Using this information may, in the future, help develop more effective antibiotics.
References:
- F. Wylie, Australian Life Scientist 9, 36 (2012).
- D. I. Fernandez, J. D. Gehman and F. Separovic, Biochim. Biophys. Acta 1788, 1630 (2009).
- D. I. Fernandez, A. P. Le Brun, T. H. Lee, P. Bansal, M. I. Aguilar, M. James, F. Separovic, Eur. Biophys. J. in press (2012). DOI: 10.1007/s00249-012-0796-6
- D. P. Tieleman, H. J. C. Berendsen, J. Chem. Phys. 105, 4871 (1996).